Conventional technology has been described in patent reference 1 (Patent application No. 2001-325773). An overview of the rotary annulus illumination type total reflection optical system, will be described below.
The Köeller optics system that illuminates an object by introducing laser beam into a peripheral region of an objective lens of a microscope is characterized by laser beam whose illumination direction is rotatable while the illumination direction is inclined with respect to the axial center of the objective lens of the microscope. The illumination system of said rotary annulus light, total internal reflection illumination mechanism introduces laser beam into the objective lens of a microscope at the peripheral region to enable total internal reflection and excite fluorescent molecules by an evanescent field that is formed by total internal reflection. Said mechanism is also provided with a mirror that has a mechanism to finely adjust the inclination of the mirror to set the incidence of the laser beam at an optimal angle for the objective lens used. It also features a means to rotate the reflector. The rotating means, including mirror, is structured with a symmetrical shape and weight about the center of a rotation axis to prevent the adverse effect of vibration during reflective rotation of said mirror on the microscope.
The principle of total reflection illumination (evanescent illumination) that makes laser beam incident upon a peripheral region of an objective lens of a microscope so as to produce fluorescence using an evanescent field is briefly described below referring to FIG. 6.
In FIG. 6, 1 denotes the objective lens, 2 immersion oil, 3 a cover glass and 4 aqueous solution. Total internal reflection occurs on an interface between the glass and the aqueous solution when the laser beam is introduced into the objective lens 1 at the peripheral region (normally 61-degree inside and 68-degree outside angles). Rays appear at approximately 150 nm from the interface (the light filed is called an evanescent field). It is possible to significantly reduce background light and derive a high-contrast single fluorescent molecule image by using this evanescent field for fluorescent illumination.
The conventional technology described above can generate high-contrast single fluorescent molecule image but it is not possible to detect the orientation, that is, the absorption transition moment, of a single fluorescent dye molecule. The absorption transition moment of a fluorescent dye is the direction in which the probability of the molecule being excited is the highest, which is determined by the molecular structure of the fluorescent dyes. The intensity of fluorescence emitted from the fluorescent dyes reaches the maximum level when the direction of polarization of the excitation light coincides with absorption transition moment.
When the light incident on the aqueous solution 4 via the cover glass (3) comprises p-polarized light, the resultant evanescent field has a transverse (X-axis) component (2) and a longitudinal (Z-axis) component (1) as shown in FIG. 7. When s-polarized light is incident, the evanescent field comprises only perfect transverse waves (a Y-axis component) as shown in FIG. 8. (Refer to Kunio Tsuruta, Applied Optics I [Applied Physical optics Series 1] p37-42, Total internal reflection and Evanescent Waves, for a detailed description.)
The absorption transition moment of fluorescent dyes is detected by rotating about the Z-axis the transverse component arrayed on the X-Y plane in the evanescent field. The intensity of the longitudinal component does not contribute to the detection of the absorption transition moment because it does not vary due to the rotation. When p-polarized light is incident, the evanescent field has both transverse and longitudinal components with decreasing intensity for the transverse component. On the other hand, when s-polarized light is incident, the resultant evanescent field has only the transverse component, and this makes it possible to efficiently detect the absorption transition moment of the fluorescent dye. When a single fluorescent dye molecule is firmly conjugated to a single molecule of an observation sample, such as a protein or DNA by covalent bond, change in the internal structure of the orientation of the sample or change of the whole can be detected as change of the orientation of the absorption transition moment at a single molecule level.
To generate such an evanescent field containing only a transverse component (i.e., oscillation in the Y-axis direction), s-polarized light oriented perpendicular to the radial direction from the optical axis (meridional plane) (reference numbers 5 and 6 in FIG. 6) must be incident on and reflected from the objective lens. If polarized rays are simply incident on the objective lens when a polarizer is rotated, not only s-polarized but also p-polarized light will be incident on the interface between the cover glass 3 and the aqueous solution 4. The resultant evanescent field will contain both longitudinal (Z-axis) and transverse (X-Y axis) components. The longitudinal (Z-axis) component does not contribute to the detection of the absorption transition moment of fluorescent dye since the longitudinal (Z-axis) component remains constant irrespective of a position of the incident laser beam. Although the transverse (X-Y axis) component rotates about the Z-axis when the position of the incident beams change, the intensity of the beam decreases because of the presence of the longitudinal component, thereby making it difficult to detect the absorption transition moment efficiently. It is difficult for the conventional simple polarized illumination system described above to detect the orientation of the absorption transition moment of fluorescent dyes.
The polarized total reflection illumination optical system by rotary annulus light of the present invention is to detect the orientation of the absorption transition moment of fluorescent dye by introducing s-polarized light oriented perpendicularly to the direction of the radiation from the center of the optical axis of the objective lens (meridional plane), and by forming an evanescent field containing only a transverse component by turning the incident rays about the optical axis of the objective lens at a low speed.
When a single fluorescent dye molecule is firmly coupled by covalent bond to a single molecule of an observation sample, such as a protein or DNA, the change in the internal structure and the orientation of the whole sample can be detected as change of the orientation of the absorption transition moment of fluorescent dye at a single molecule level.
The conventional system described above lacks the means to convert the annulus rays incident on the objective lens into the shape of a true circle (e.g., by means of the addition of a beam-correction prism). When parallel beam is incident at 45 degrees from the rotational center of a rotary reflector, the reflected orbicular rays are elliptic in shape. To form the beam into a true circle shape, an anamorphic prism pair which is used for semiconductor laser beam-shaping may be used.
The present invention offers annulus illumination rays of a true circular shape to achieve ideal evanescent illumination in which the primary and secondary diffraction of light, which is the major cause of noise, is removed.